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. 2019 Sep 26;9(10):373. doi: 10.1007/s13205-019-1904-4

Biodegradation of NSAIDs and their effect on the activity of ligninolytic enzymes from Pleurotus djamor

Rosbi Cruz-Ornelas 1, José E Sánchez-Vázquez 1, Lorena Amaya-Delgado 2, Karina Guillén-Navarro 1, Angeles Calixto-Romo 1,✉
PMCID: PMC6763541  PMID: 31588397

Abstract

In this work, the white-rot fungus Pleurotus djamor was used for the first time to determine the degradation kinetics of the nonsteroidal anti-inflammatory drugs diclofenac, naproxen and, ketoprofen, either individually or in mixtures, in submerged cultures. Removal of 93% individual diclofenac and 99% diclofenac in mixtures with naproxen and ketoprofen at 6 h of incubation with the fungus was achieved. The elimination levels of naproxen and ketoprofen individually were 90% and 87%, respectively, after 48 h of incubation. However, the removal levels of these compounds in mixtures were 85% and 83%, respectively. On the other hand, during the degradation kinetics analysis, the enzymatic activities of laccases, manganese peroxidases, and lignin peroxidases were evaluated, yielding values of 3700, 270 and 31 U/L, respectively. Additionally, it was demonstrated that during degradation of diclofenac or the three drugs mixed in the submerged cultures, the enzymatic activity of extracellular laccases expressed by P. djamor increased by 200% and 300%, respectively. The activity of manganese peroxides increased by 126% with diclofenac and 138% when the mixture of drugs was added to the cultures. On the other hand, lignin peroxidase only increased activity by 123% with the drug mixture.

Keywords: Pleurotus djamor, White-rot fungus, Laccases, Manganese peroxidase, Lignin peroxidase

Introduction

Among the different classes of pharmaceutical products used in human and veterinary medicine, are nonsteroidal anti-inflammatory drugs (NSAIDs), a group of drugs commonly used throughout the world because they have analgesic, antipyretic and anti-inflammatory effects. Many ingested NSAIDs are not metabolized at 100%; a high percentage is excreted through urine and feces (Ankley et al. 2007) and released into wastewaters. Additionally, expired or unused products, such as those in municipal garbage dumps, rivers, toilets or municipal wastewater treatment plants, have inadequate final disposal. The concentrations of NSAIDs in the aquatic environment are not high, but bioaccumulation of these drugs causes undesired effects on aquatic fauna, as was demonstrated in fishes (Yang et al. 2013).

The most commonly used NSAIDs are diclofenac, naproxen, and ketoprofen. Ecotoxicological studies have been conducted in different aquatic environments, finding that diclofenac causes damage to aquatic organisms (Eades and Waring 2010; Quinn et al. 2011; Feito et al. 2012; Mezzelani et al. 2016). This damage to flora and fauna has led to the inclusion of diclofenac in the list of substances that should be evaluated in wastewater in Europe according to the European Water Framework Directive (Ribeiro et al. 2015). In addition, diclofenac and ketoprofen used in veterinary medicine have caused unfavorable effects on vulture populations through the food chain, causing high mortality associated with renal failure and visceral gout (Naidoo et al. 2010; Martínez et al. 2013). On the other hand, some reports show that a constant discharge of naproxen into the aquatic environment has caused chronic effects on aquatic organisms such as crustaceans and algae (Fernández et al. 2010). NSAIDs can be found together with other drugs, such as antibiotics, analgesics, and contraceptives, among others, which can cause other toxic or harmful effects on aquatic flora or fauna (Yang et al. 2013). Currently, conventional treatments used by most wastewater treatment plants only result in the partial degradation of some drugs because most of them have complex structures, low bioavailability and are bioaccumulative (Stackelberg et al. 2004; Caliman and Gavrilescu 2009). For these reasons, researchers around the world are searching for different degradation processes of NSAIDs, which are considered as emerging pollutants.

During recent decades, physicochemical and biological treatments for the elimination of NSAIDs in the environment have been evaluated. For example, the removal of diclofenac and naproxen by coagulation–flocculation and flotation processes (Suarez et al. 2009), and the degradation of naproxen and ketoprofen by anaerobic digestion (Carballa et al. 2007) eliminate 40 and 50% of each drug, respectively. Advanced oxidation processes (AOPs) have been carried out with better results, such as ozonolysis (Jankunaite et al. 2017), electro-Fenton and anodic oxidation (Feng et al. 2014; Martínez et al. 2013; Jankunaite et al. 2017) and photocatalysis systems (Martínez et al. 2013; Jankunaite et al. 2017), which have obtained degradation levels between 80 and 100% for diclofenac, naproxen, and ketoprofen. However, the cost of their application and the secondary contamination produced by some methods due to the use of chemical products led to the search for alternatives to eliminate microcontaminants in different aquatic environments. For these reasons, it is necessary to search for better systems of degradation to achieve mineralization and lower process costs without causing secondary pollution, as in the biological systems. Several bioremediation processes use white-rot fungi (WRF) due to their ability to degrade a wide variety of xenobiotics through their extracellular oxidative enzymes (Vasiliadou et al. 2016; Shreve et al. 2016).

The WRF are able to degrade a wide variety of organic compounds, such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls or organochlorine pesticides (Vandertol-Vanier et al. 2002; Cvancarová et al. 2012), until its mineralization through the synergistic action of an enzymatic system of oxidoreductases as lignin peroxidase, manganese peroxidase, versatile peroxidase, and laccase. The mechanism of lignin-degrading enzymes is based on the production of free radicals that allow these enzymes to be catalytically active on a variety of organic compounds. Thus, WRF have been used to perform degradation studies of recalcitrant contaminants in soils or wastewater. Most studies have evaluated Trametes versicolor because it has greater laccase activity than Pleurotus spp. However, when used in nonsterile environments, Pleurotus ostreatus maintains its enzymatic activity longer (16 weeks) than T. versicolor (Jonas 2015). For these reasons, it is important to evaluate other organisms of the Pleurotus genus in soil and water bioremediation processes.

P. djamor is a white-rot fungus widely distributed in Mexico and is considered as the most important pantropical species of the genus Pleurotus spp. This fungus has the capacity to express lignin-cellulolytic enzymes during its life cycle, mainly laccases (Moreno et al. 2014). P. djamor has been studied in bioremediation processes as a degrader of endosulfan using solid-medium cultures of P. djamor; also, it has been demonstrated as an atrazine-tolerant species (Chan-Cupul et al. 2014). This fungus has also been used in the removal of dyes  from the textile industry (Alonso-Calderon et al. 2017) and in the removal of organic matter measured as chemical oxygen demand (COD) (Yildirim et al. 2012).

However, P. djamor has not been studied for the degradation of NSAIDs or to determine the effect caused by NSAIDs on laccase, manganese peroxidase or lignin peroxidase activities. For this reason, in the present work, the degradation of diclofenac, naproxen and ketoprofen by P. djamor strain ECS-0123 was evaluated either individually or in mixtures, and the enzymatic activities of laccases, manganese peroxidases, and lignin peroxidases were evaluated over 8 days in the presence of NSAIDs.

Materials and methods

Chemical products

The pharmaceutical compounds diclofenac, naproxen and ketoprofen were obtained from Sigma Aldrich. The methanol used in chromatography was HPLC-grade (J.T. Baker). A standard solution of each NSAID (1000 mg/L) dissolved in ethanol (J.T. Baker) was prepared.

Culture of Pleurotus djamor

The fungus P. djamor (ECS-0123) was obtained from the mycological collection of ECOSUR, Tapachula, Chiapas, Mexico. The culture medium was used for the growth of the fungus (liquid or solid) comprised malt extract (12 g/L), glucose (10 g/L), and peptone (1 g/L), pH 6.4; bacteriological agar (12 g/L) was added when solid medium was used. The strain was propagated in solid medium in darkness at 25 ± 1 °C for 7 days. After the growth time, five mycelial fragments (5 mm diameter) were collected and placed in 30 mL of liquid medium in 125-mL Erlenmeyer flasks, which were kept at 25 ± 1 °C at 120 rpm for 5 days of growth in darkness. Then, the mycelia and the supernatant were homogenized under aseptic conditions in a mixer for 30 s. Once the homogenized suspension was obtained, one mL of the culture was taken to inoculate 30 mL of liquid medium in Erlenmeyer flasks. P. djamor was grown at 25 ± 1 °C at 120 rpm in darkness.

Kinetics of degradation of pharmaceutical compounds

After the fifth day of P. djamor growth observed as pellets, NSAIDs were added to the cultures individually (10 mg/L each) or as a mixture of the three drugs (7 mg/L each). Drugs degradation was reached out on a shaker (120 rpm) at 25 ± 1 °C in darkness. Two controls were included in the degradation assays using the culture medium from Sect. "Culture of Pleurotus djamor": Control 1 without inoculation and control 2 with fungi inactivated at 121 °C for 15 min. The degradation of the pharmaceutical compounds was determined at 0.25, 0.5, 1, 3, 6, 9, 24, 48, and 72 h. Assays were evaluated three times with three replicates each one.

Determination of pharmaceutical compounds by LC–MS/MS

One milliliter of sample was taken at each time point and filtered through a Millex-GV (Millipore) 0.22-μm filter and subsequently analyzed. The drugs were determined in a liquid chromatograph coupled to a triple quadrupole mass spectrometer (Agilent Technologies, Mod. 64109). The mobile phase used was a mixture of methanol and deionized water (80:20) with a flow rate of 0.7 mL/min, and chromatographic separation was achieved by a Zorbax Eclipse XDB C-18 (4.6 × 150 mm, 5 µm). The sample volume injected was 1 μl, and the ionization of the samples was performed in electrospray ionization mode with negative polarity. The retention times were 5, 6 and 7 min for ketoprofen, naproxen and diclofenac, respectively. The limit of detection for NSAIDs was 0.1 mg/L.

Determination of enzymatic activities

The enzymatic activities of laccase, manganese peroxidase, and lignin peroxidase were determined every 24 h for 8 days. One control was included in the enzymatic assay, of each condition which consisted of using culture medium without NSAIDs. Each assay was evaluated three times with three replicates. A UV-1240 UV–VIS spectrophotometer was used for determining absorbance (Shimadzu, Inc., Japan).

Laccase activity

The activity was determined using 0.5 mM 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate in 0.1 M acetate buffer, pH 5. The reaction mixture was incubated at 25 °C for 3 min, and the increase in absorbance was measured at 420 nm using a molar extinction coefficient of 3600 mM−1 cm−1 (Moreno et al. 2014). One unit (U) of laccase activity is defined as the amount of enzyme required to oxidize 1 μmol of ABTS for 1 min under the specified conditions.

Manganese peroxidase activity

The activity was carried out in a reaction mixture with 0.1 mM MnSO4, 0.1 mM H2O2 and enzymatic extract in 0.1 M sodium tartrate buffer, pH 5 at 30 °C for 5 min. The reaction was initiated by the addition of H2O2. The oxidation of Mn2+ to Mn3+ was measured at a wavelength of 240 nm using a molar extinction coefficient of 6500 mM−1 cm−1 by increasing the absorbance. One unit (U) of MnP activity was defined as the amount of enzyme required to oxidize 1 μmol of Mn2+ to Mn3+ per minute under the specified conditions.

Lignin peroxidase activity: Lignin peroxidase activity was measured using veratryl alcohol at 2 mM, 0.4 mM H2O2 and enzymatic extract in 0.1 M sodium tartrate buffer, pH 3. The reaction was initiated when H2O2 was added and then incubated at 30 °C for 10 min. The oxidation of veratryl alcohol was measured by increasing the absorbance at a wavelength of 310 nm using a molar extinction coefficient of 9300 mM−1 cm−1. One unit (U) of LiP activity was defined as the amount of enzyme required to oxidize 1 μmol of veratryl alcohol for 1 min under the specified conditions.

Results and discussions

Degradation of pharmaceutical compounds

In this study, P. djamor cultures in the form of pellets were used for the degradation of diclofenac, naproxen, and ketoprofen individually or as a mixture of the three NSAIDs without mediators or inducers, showing that P. djamor was able to degrade the drugs in any conditions (Fig. 1). After the addition of diclofenac in P. djamor cultures, a degradation of 82% was observed during the first 15 min, less time than degradation reached out in other reports (Rodarte-Morales et al. 2012; Cruz-Morató et al. 2014; Marco-Urrea et al. 2010a, b); see Table 1. According to the results, 93% elimination of diclofenac and 90% elimination of naproxen were achieved at 6 and 72 h, respectively (Fig. 1a, b). These results show high percentages of degradation compared with anaerobic digestion processes, where they only reach 46% (Carballa et al. 2007) or 42% in the coagulation–flocculation method (Suarez et al. 2009).

Fig. 1.

Fig. 1

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Kinetics of NSAID degradation with the fungus P. djamor. a Diclofenac (DCF); b naproxen (NPX); c ketoprofen, (KTP); d mixture of the three NSAIDs. The bars indicate the standard error of three assays with three replicates each

Table 1.

Percentages of degradation of diclofenac, naproxen, and ketoprofen compared with other works

Organism NSAID Degradation (%) Degradation time (h) References
Phanerochaete chrysosporium

Diclofenac

Naproxen

 100 96 Rodarte-Morales et al. (2012)
Bjerkandera adusta

Diclofenac

Naproxen

 100 168 Rodarte-Morales et al. (2012)
Trametes versicolor Diclofenac 94 1 Marco-Urrea et al. (2010b)
Naproxen 100 6 Marco-Urrea et al. (2010a)
Anaerobic digester Diclofenac 69 480 Carballa et al. (2007)
Naproxen 88 480 Carballa et al. (2007)
Trametes versicolor Ketoprofen 100 48 Cruz-Morató et al. (2014)
Trametes versicolor Ketoprofen 94 2160 Nguyen et al. (2013)
Trametes versicolor Ketoprofen 99 24 Marco-Urrea et al. (2010c)
Pleurotus djamor Diclofenac 98 6 This work
Naproxen 90 72
Ketoprofen 85 72
Pleurotus djamor Diclofenac 99 48 This work
Naproxen 87 48
Ketoprofen 83 48
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In contrast, the degradation of naproxen and ketoprofen was slower, resulting in 53% for naproxen and 42% for ketoprofen (Fig. 1 b, c) during the first 12 h and 90% and 83% at 72 h, respectively.

On the other hand, there are few reports on the degradation of ketoprofen, some of which have evaluated anaerobic digestion processes that obtained 50% elimination of the drug (Carballa et al. 2007). Recently, some AOPs have been applied in the degradation of ketoprofen, such as photocatalysis, electro-Fenton oxidation and anodic oxidation, and have achieved degradation of 60% or complete elimination of the drug in the first 2 h (Feng et al. 2014). In contrast, Marco-Urrea et al. (2010c) studied the degradation of ketoprofen (10 mg/L) with T. versicolor in a defined medium, reaching 99% degradation in 24 h. Cruz-Morató et al. (2014) and Nguyen et al. (2013) evaluated the degradation of ketoprofen at two concentrations (0.8 μg/L and 5 μg/L), achieving total elimination in 2 days for the first concentration and 94% degradation in 90 days for the second concentration. Moreover, in this work, 85% ketoprofen elimination was reached in 72 h (Fig. 1c).

Considering that a large number of micropollutants are found in aquatic environments and form complex mixtures, we evaluated the degradation of diclofenac, naproxen, and ketoprofen mixtures in this work. After 6 h of adding the drug mixture to fungal cultures, 99, 72 and 68% degradation levels for diclofenac, naproxen, and ketoprofen, respectively, were obtained. At 48 h, of 99% for diclofenac, 87% for naproxen and 83% for ketoprofen degradation were obtained. It is worth mentioning that the pH of the culture media was measured after degradation assays obtaining a value of 4.6 each replicate.

Diclofenac was eliminated at a higher proportion than the other drugs during the first hours. This can be attributed to its amino group, which acts as an electron donor and is a highly reactive functional group. This drug also contains chlorine, which is considered as a strong electron acceptor because it belongs to the halogen group. These functional groups could be the possible causes of higher diclofenac degradation in short times. In contrast, naproxen and ketoprofen contain carboxyl groups, which act as electron acceptors, and ether and ketone act as electron donor groups; these are less reactive than the amino groups from diclofenac, explaining the lower removal levels of naproxen and ketoprofen.

On the other hand, controls containing heat-inactivated P. djamor reduced a small proportion of the NSAIDs in the culture medium (Fig. 1). This effect could be similar to inactive, thermally treated cells of T. versicolor and P. sajor-caju, which showed an alteration in their physiochemical properties, such as acquiring a bioadsorptive affinity for mercury ions (Arica et al. 2003). Living cells possess negatively charged phospholipid membranes, thus avoiding adsorption to negatively charged molecules, such as the drugs studied in this work. Therefore, the degradation of the drugs is attributed mainly to the action of the enzymes produced by P. djamor and not because of bioadsorption. On the other hand, the pH measured during the degradation assay was 4.6, and this condition could increase the removal of the NSAIDs as previously described in other works; for example, an acidic environment promotes removal of acidic pharmaceutical compounds by biological systems (Urase and Kikuta 2005). In the negative controls without P. djamor, no decrease in the drugs was observed (Fig. 1).

Enzymatic activity

The biodegradation of drugs such as NSAIDs by WRF is attributed to the presence of extracellular and intracellular enzymes produced by the fungi which act by oxidative mechanisms intracellularly or extracellularly (Vasiliadou et al. 2016). However, not all WRF species produce the same ratio of extracellular ligninolytic enzymes (laccase, manganese peroxidase, versatile peroxidase or lignin peroxidase). Furthermore, the combination of enzymes produced varies from one species to another, which can cause different rates of biodegradation of the same compound with different WRF. Additionally, most degradation studies do not take into account the activity of the different oxidoreductases expressed by the WRF. Likewise, fungi cultivation conditions, such as aeration or the composition of the culture medium, can influence the efficiency of degradation. For these reasons, ligninolytic enzymes expressed by P. djamor were analyzed in this work.

Laccase activity increased 200% when diclofenac was added and 300% when the drug mixtures were added to the cultures. Additionally, we observed an increase of 25% when naproxen or ketoprofen was added individually (Fig. 2a). The activity of manganese peroxides increased 126% with diclofenac and 138% when the mixture of drugs was added to the cultures (Fig. 2b). Mean while lignin peroxidase only increased activity by 123% with the drug mixture (Fig. 2c). The expression of laccase, lignin peroxidase, and manganese peroxidase is often induced by various physiological factors. For example, its promoter region may contain elements that participate in metals response mechanism (MRE, metal response element), in xenobiotics response (XRE, xenobiotic response element), heat shock element response (HSE, heat shock element) or response to oxidative stress (ARE, antioxidant response element) (Janusza et al. 2013). In addition, there are some factors, such as pH, that favor or suppress the expression of some enzymes. In this study, the pH measured during the degradation tests showed a slightly acidic character of 4.6 which could have favored the expression of laccases but not that of the manganese peroxidases enzymes which are repressed in the presence of acidic environments (Fernández-Fueyo et al. 2014). On the other hand, it is known that aromatic compounds structurally related to lignin or lignin derivatives can increase the induction of laccases at the transcriptional level and it has been found that the effects differ according to the aromatic compounds analyzed, resulting in the expression of different isoenzymes in the same organism (Zhuo et al. 2017).

Fig. 2.

Fig. 2

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Extracellular enzymatic activity of laccase (a), manganese peroxidase (b), and lignin peroxidase (c) of P. djamor during the degradation of drugs (T 25 °C, 120 rpm, pH 4.6). The bars indicate the standard error of three assays with three replicates each one

The chemical structure of the NSAIDs used in this work belongs to the group of aromatic compounds which could have favored the increase of enzymatic activities, especially laccases (Fig. 2a). In addition, when there is more than one aromatic compound, the enzymatic activity of laccases increases its activity due to a synergistic effect of this type of compounds (Zhuo et al. 2017).

During the degradation of mixed NSAIDs by P. djamor, the presence of laccase, manganese peroxidase, and lignin peroxidase was determined, and their enzymatic activities were measured. Maximum activities of 3668, 270 and 31 U/L, respectively, were found after 8 days of incubation with the drugs. These activities are considerably higher than those observed by other authors with T. versicolor (50 and 20 U/L for laccases and manganese peroxidases, respectively) or Ganoderma lucidum (20 and 40 U/L of manganese peroxidase activity) (Vasiliadou et al. 2016). These determinations were performed after 8 days of incubation with the drugs. In the case of lignin peroxidase, the maximum activities of 50 U/L and < 10 U/L for T. versicolor and G. lucidum, respectively, were reached at the 2nd and 3rd days of incubation with the drug mixture.

Conclusions

In this study, we revealed for the first time the positive effect of NSAIDs (diclofenac, naproxen, and ketoprofen) on the activity of three oxidoreductases expressed by P. djamor, while degradation of these NSAIDs is accomplished. The presence of diclofenac increased the enzymatic activity of laccase, which improved twofold when diclofenac was added individually and threefold when mixtures of the three drugs were added. These results show the potential application of this fungus for future studies of the degradation of other NSAIDs and other types of emerging pollutants or compounds with a similar chemical structure to those studied in this work, especially emerging mixtures of contaminants.

Acknowledgements

RCO is grateful for the scholarship given by the Consejo Nacional de Ciencia y Tecnología (CONACyT). We thank Lilia Moreno for her technical support in the fungal cultures and biochemical assays.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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